G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors that represent the highly desirable drug targets for diverse diseases, including cancer. Despite their importance, our understanding of how GPCRs contribute to tumorigenesis and cancer progression remains limited, because there are over 350 non-sensory GPCRs and many of them are aberrantly expressed in cancer cells. This proposal aims to delineate the function and mechanisms of a subgroup of GPCRs, Gi/o-GPCRs, in regulating the self-renewal and tumorigenic activities of stem cell-like breast tumor-initiating cells (TICs). We will test the idea that targeting Gi/o-GPCRs can eliminate TICs and restore patient response to current therapeutics. As a model of our studies, we focus on ErbB2/HER2-positive breast cancers, because they are highly aggressive and have poor prognosis. HER2-targeted therapy is the preferred treatment for these cancers but drug resistance is the major hurdle. The mechanisms underlying drug resistance remain largely unknown but are attributed to a reservoir of TICs in tumors. TICs may escape therapy in part because they find ways to transactivate HER2 via other pathways?including pathways mediated by GPCRs. Our preliminary studies found Gi/o-GPCRs were overexpressed in human HER2-amplified breast cancer cell lines and mouse TICs from HER2-driven mammary tumors, and were required for growth of TICs in vitro and in vivo. Using our transgenic mouse lines that allow us to upregulate or block Gi/o signaling, in an inducible manner, specifically in mammary epithelial cells, we further showed HER2-driven spontaneous formation of mammary tumors and lung metastases requires Gi/o-GPCR signaling. These exciting data led to our proposal's central hypothesis: HER2-driven breast tumors harbor a reservoir population of TICs that upregulate Gi/o-GPCR signaling; and this hyperactivates EGFR/HER2, permitting TIC self-renewal and ultimately, promoting tumor growth and metastasis. We propose that disrupting Gi/o-GPCR signaling might be a way to ablate TICs, prevent resistance, and maintain effectiveness of HER2-targeted therapeutics . Our proposal uses multimodal approaches, including genetic and patient-derived xenograft mouse models of breast cancer. With this powerful toolkit we will determine the mechanisms of upregulated Gi/o signaling in TICs to increase TIC tumorigenicity and drive cancer development (Aim 1); elucidate if Gi/o- GPCRs function in part through transactivation of EGFR/HER2 via the activation of G?i/o/c-Src and G??-PI3K? signaling axes to drive cancer progression (Aim 2); and finally, test whether blocking Gi/o-GPCR signaling can eliminate TICs and enhance sensitivity to current treatment regimens for HER2+ breast cancer (Aim 3). The overall impact of our studies will be a fundamental advance in our mechanistic understanding of how tumorigenic TICs acquire the ability to transactivate EGFR/HER2 via Gi/o-GPCR signaling. This knowledge may be exploited for finding new ways to eliminate TICs to augment HER2 therapeutics.